Analytical monitoring of the production of biodiesel by high-performance liquid chromatography with various detection methods

Abstract

Gradient elution reversed-phase high-performance liquid chromatography (RP-HPLC) was used for the determination of compounds occurring during the production of biodiesel from rapeseed oil. Individual triacylglycerols (TGs), diacylglycerols, monoacylglycerols and methyl esters of oleic, linoleic and linolenic acids and free fatty acids were separated in 25 min using a combined linear gradient with aqueous–organic and non-aqueous mobile phase steps: 70% acetonitrile+30% water in 0 min, 100% acetonitrile in 10 min, 50% acetonitrile+50% 2-propanol–hexane (5:4, v/v) in 20 min and 5 min final hold-up. Another method with a non-aqueous linear mobile phase gradient [from 100% methanol to 50% methanol+50% 2-propanol–hexane (5:4, v/v) in 15 min] was used for fast monitoring of conversion of rapeseed oil triacylglycerols to fatty acid methyl esters and for quantitation of residual TGs in the final biodiesel product. Sensitivity and linearity of various detection modes (UV detection at 205 nm, evaporative light scattering detection and mass spectrometric detection) were compared. The individual sample compounds were identified using coupled HPLC–atmospheric pressure chemical ionization mass spectrometry in the positive-ion mode.

Introduction

In the last decade, biodiesel has been introduced as an environmentally more friendly substitute for fossil diesel fuels. Biodiesel is produced from renewable sources by transesterification of triacylglycerols (TGs) of fatty acids in vegetable oils (e.g., rapeseed, sunflower or soybean oil) to methyl esters (MEs) of fatty acids [1]. The presence of even small amounts of the original unconverted oil compounds in biodiesel can cause engine problems and results in increased production of hazardous emissions [2]. Hence, a sensitive and reliable analytical method is needed to monitor the contents of TGs, diacylglycerols (DGs) and monoacylglycerols (MGs) in biodiesel. Maximum allowed concentration limits are specified in the national standards, e.g., in the Czech Republic (0.24%) [3], Austria [4], Italy [5], Germany [6] and France [7] and will be included in the European standard specifications based on the EN 590 for fossil diesel fuels [8], [9], [10].

Gas chromatography (GC) can be used for the determination of MEs, but it is less convenient for the analysis of non-volatile acylglycerols, which need to be derivatized before the analysis by trimethylsilylation [11], [12], [13] or acetylation [14] of the free hydroxyl groups in MGs and DGs. GC determination of underivatized TGs is feasible at high column temperatures (approximately 350°C) and requires a short capillary column with a good temperature stability [14], [15]. On-line coupling of normal-phase high-performance liquid chromatography (HPLC) with GC was employed for the determination of acetylated MGs and DGs and of TGs in biodiesel [14]. GC was also used for the determination of residual glycerol in biodiesel after derivatization [16]. Screening analysis of unknown seed oils with attention to GC and GC–mass spectrometry (MS) of fatty acid derivatives and methods for the determination of the configuration of double bonds in the fatty acids were reviewed by Spitzer [17]. GC methods for the analysis of TGs usually utilize volatile derivatives of fatty acids [17], [18], but a large number of derivatives suitable for HPLC with UV or fluorescence detection was also proposed [18].

HPLC makes feasible direct analysis of all biodiesel components without derivatization. However, acylglycerols and methyl esters do not absorb in the UV region at wavelengths higher than 220 nm, which causes detection problems. Various detection techniques have been employed for the determination of TGs to substitute conventional UV detection [19], [20], including the use of moving wire detection [21], density detection [22], flame ionization detection (FID) [23], refractive index detection [24], [25], [26], [27], evaporative light scattering detection (ELSD) [28], [29], [30], [31], [32], [33], [34] and mass spectrometric detection [35], [36], [37]. Non-aqueous reversed-phase (NARP) HPLC has become the established technique for the separation of TGs, both in the isocratic [31], [32], [33], [35] and in the gradient elution mode [20], [23], [33], [34], [36], [37], [38], [39], [40]. NARP-HPLC with acetonitrile–dichloromethane (68:32) allows distinguishing between the TGs with the same molecular masses and different position of the double bonds, e.g., LLL vs. OLLn [31], [32] (see Table 1 for notations). With 100% propionitrile as the mobile phase [35] it is possible to separate individual TGs and DGs differing in the equivalent carbon number (ECN), which is defined as ECN=CN−2DB, where CN is the number of carbon atoms in the acyl chains of acylglycerol and DB is the number of double bonds. In NARP-HPLC with a linear gradient from 100% methanol to 100% 2-propanol, the retention times of TGs can be predicted [41] on the basis of the experimental retention times of various synthetic standards [20].

Silver-phase HPLC has been often used for separation of lipids differing in the number and position of double bonds [42], [43], [44]. This separation technique was successfully coupled with atmospheric pressure chemical ionization (APCI) MS [45] or with electrospray ionization (ESI) MS [46]. Silver ions form weaker complexes with γ-linoleic acyls than with α-linoleic acyls in TGs. Further, separation of isomeric TGs SOO from OSO and SSO from SOS (where S=saturated fatty acid and O=monounsaturated fatty acid) was possible with silver-phase HPLC [46]. In addition to silver-phase HPLC, Christie reviewed also the applications of chiral HPLC to the structural analysis of TGs. Chromatography on a strong cation-exchange column in the silver ion form was used to separate and determine eight geometrical isomers of the phenacyl esters of linolenic acid [47].

In addition to RP-HPLC, normal-phase chromatography on a cyanopropyl silica column can separate partially transesterified lipids into fractions containing lower alkyl esters, free fatty acids, TGs, 1,3-DGs, 1,2-DGs and MGs [30]. Similar group separation, but without distinguishing between 1,3- and 1,2-diacylglycerols, was reported using a cyanopropyl silica column coupled with two gel-permeation chromatographic columns [22]. Normal-phase HPLC was used for the separation of TGs according to the number of double bonds with partial separation according to the acid chain length, the esters with longer chains being eluted first [48]. However, normal-phase chromatography is not suitable for the separation of the individual compounds in the same ester class.

Sandra et al. [49] compared separations of TGs using microcolumn HPLC and non-aqueous capillary electrochromatography (CEC) with 10 mM ammonium acetate in acetonitrile–2-propanol–hexane (57:38:5) as the mobile phase. CEC provided better resolution of TGs differing in the position of double bonds (e.g., LLL from OLLn) than HPLC.

MS with various ionization techniques has been successfully used for structure determination of lipids. Barber et al. [50] suggested the fragmentation pattern of TGs using electron ionization (EI). Later, the positive-ion mode chemical ionization (CI) with ammonia was applied for the structure analysis of diacyl phosphatidylcholine [51] and CI in the negative-ion mode for the analysis of TGs [52], [53]. CI is a softer ionization technique than EI and the CI mass spectra yield protonated molecular ions of diacyl phosphatidylcholine in contrast to EI. More recent ionization techniques, like thermospray ionization (TSI), ESI and APCI are superior to EI and CI in the field of the lipid analysis for the following reasons: (1) the molecular mass determination is easier; (2) the structural information can be also obtained, as the fragmentation process can be enhanced by collisionally induced dissociation (CID) in-source or in the MS–MS arrangement; (3) the possibility of direct coupling of soft ionization MS with HPLC makes feasible the analysis of complex mixtures of natural lipids. ESI is the softest ionization technique as it yields only the protonated molecular ions of TGs with no fragmentation, so that an MS–MS instrument is necessary to obtain structural information on the acyl chains [54]. ESI-MS–MS was used to localize the positions of double bonds of polyunsaturated fatty acids [55]. However, the performance of ESI is not optimum with non-aqueous mobile phases. Both molecular mass and structural information on phospholipids and related compounds was obtained with TSI [40]. A very efficient method for the structure elucidation of TGs using ESI- and fast atom bombardment (FAB) MS–MS has been reported recently [56].

Various HPLC–MS methods for the lipid analysis have been recently reviewed [57], [58]. The most successful ionization technique in this field is probably APCI [35], [36], [37], [59], which provides a sensitive HPLC detection, both the structural and the molecular mass information and full compatibility with common HPLC conditions for the separation of TGs. APCI-MS was also applied for the analysis of TGs containing hydroxy [60], hydroperoxy [61] and epoxy [37] groups.

Supercritical fluid chromatography (SFC) coupled with EI-MS was applied for the characterization of the butter fat TGs [62] and more recently SFC–APCI-MS has elucidated information on both the molecular masses and on the acyl chain composition in TGs [63], [64]. The relative abundance of the [M+H-R_iCOOH]⁺ ion makes possible distinguishing between α- and γ-linoleic residues in the same regiospecific position on the glycerol backbone [64].

In the present work, UV detection, ELSD and APCI-MS are compared with respect to their sensitivity, compatibility with gradient elution and possibility of peak identification of TGs without authentic standards. To our knowledge, these three techniques for HPLC detection of TGs have not been compared so far. The objective of the present work is the development of an HPLC method suitable for the separation of TGs, DGs, MGs and MEs occurring during the production of biodiesel from the rapeseed oil. According to the literature [8], TGs in rapeseed oil contain mainly oleic (60%), linoleic (20%) and linolenic (8%) acids with lower concentrations of some other fatty acids, such as palmitic (4%), gadoleic (2–3%) and stearic (1–2%) acids. We have focussed our attention on the esters of the three most abundant acids with different ECNs. The separation of TGs within the class of the same ECN is presently being investigated. Finally, the fragmentation in APCI-MS is discussed and compared with earlier published data.